Serine 937 phosphorylation enhances KCC2 activity and strengthens synaptic inhibition

The potassium chloride cotransporter KCC2 is crucial for Cl- extrusion from mature neurons and thus key to hyperpolarizing inhibition. Auditory brainstem circuits contain well-understood inhibitory projections and provide a potent model to study the regulation of synaptic inhibition. Two peculiarities of the auditory brainstem are (i) posttranslational activation of KCC2 during development and (ii) extremely negative reversal potentials in specific circuits. To investigate the role of the potent phospho-site serine 937 therein, we generated a KCC2 Thr934Ala/Ser937Asp double mutation, in which Ser937 is replaced by aspartate mimicking the phosphorylated state, and the neighbouring Thr934 arrested in the dephosphorylated state. This double mutant showed a twofold increased transport activity in HEK293 cells, raising the hypothesis that auditory brainstem neurons show lower [Cl−]i. and increased glycinergic inhibition. This was tested in a mouse model carrying the same KCC2 Thr934Ala/Ser937Asp mutation by the use of the CRISPR/Cas9 technology. Homozygous KCC2 Thr934Ala/Ser937Asp mice showed an earlier developmental onset of hyperpolarisation in the auditory brainstem. Mature neurons displayed stronger glycinergic inhibition due to hyperpolarized ECl−. These data demonstrate that phospho-regulation of KCC2 Ser937 is a potent way to interfere with the excitation-inhibition balance in neural circuits.

In most brain areas including cortex, hippocampus and the cerebellum, the D/H shift is caused by a decrease in NKCC1 and an increase in KCC2 expression during development 31,33,[40][41][42][43][44][45][46] .Interestingly, auditory brainstem neurons perinatally express KCC2 at high levels in the plasma membrane, but in a transport-inactive form [47][48][49] .Here, the developmental increase in KCC2 transport activity correlates with an increase in the oligomer/monomer ratio 49 and a switch from membrane rafts to non-membrane rafts 50 .In addition, phospho-regulation may play an essential role in the developmental activation of KCC2 51,52 , but detailed knowledge of the multiple phosphorylation sites is lacking.
In the mature auditory brainstem, synaptic inhibition is key in various computational tasks during neural signal processing.This is reflected by well-defined inhibitory projections and circuit-specific hyperpolarizing glycine reversal potentials (E Gly ).The superior paraolivary nucleus (SPN) is involved in encoding sound offsets 53 and displays an exceptionally negative E Gly = − 90.6 mV, compared to other auditory nuclei, such as the auditory lateral superior olive (LSO) (E Gly = − 68.6 mV) and the medial superior olive (MSO) (E Gly = − 75.8 mV) in mice 54 .
Here, we used CRISPR/Cas9 gene editing to generate transgenic mice with a Thr 934Ala /Ser 937Asp double mutation in KCC2 to probe for a functional link between KCC2 phosphorylation and its transport activity.

Earlier D/H shift in LSO neurons of homozygous KCC2 Thr 934Ala /Ser 937Asp mice
The action of glycine, the main inhibitory neurotransmitter in the brainstem, shows a D/H shift between P4 and P8 in LSO neurons 8,20,47,74,75 .To access whether phosphorylation of KCC2 affects its transport activity, we performed gramicidin perforated patch clamp recordings in LSO neurons at the ages P3 and P5, as previously described [76][77][78] .To this end, the reversal potential of glycine-evoked currents was determined while leaving the [Cl − ] i undisturbed through gramicidin-pores that are exclusively permeable to monovalent cations and small, uncharged molecules 79,80 .Glycine was applied via puff-application (100 ms) at saturating concentration of 1 mM, while neurons were held at different membrane potentials.Synaptic stimulation can not be used to reliably estimate E Cl− at this immature stage, due to ongoing development of inhibitory inputs 20,[81][82][83] .In control mice, we measured a depolarized E Cl− at P3 compared to P5 (Fig. 5) and hypothesized in homozygous KCC2 AD/AD mice a more hyperpolarized E Cl− with respect to controls.In the control data set containing cells from wild type (KCC2 +/+ ) littermates and C57Bl/6N animals, we recorded the average E Cl− at P3 = − 36.6 mV (n = 7) and at P5 = − 63.8 mV (n = 5), in agreement with a developmental shift toward hyperpolarizing values (**, p = 0.002).In contrast, recordings in KCC2 AD/AD LSO neurons showed the average E Cl− = − 79.4 mV (n = 5) already at P3, with no further change at P5 (E Cl− = − 71 mV, n = 5, p = 0.155).These data demonstrate that the phospho-mimetic

Adult homozygous KCC2 Thr 934Ala /Ser 937Asp mice show more hyperpolarized IPSC-reversal potentials
Based on the increased Cl − extrusion activity of KCC2 observed in HEK293 cells and immature neurons, we predicted more hyperpolarized reversal potentials for synaptically evoked inhibition (E IPSCs ) in mature LSO and SPN neurons.We tested this hypothesis by taking advantage of whole cell patch-clamp recordings that allowed to set high intracellular Cl -concentrations of 34.5 mM in order to further challenge KCC2's Cl -extrusion activity.
Based on the Nernst equation, the compositions of pipette solution, the ACSF, the junction potential correction and the assumption that glycinergic synaptic currents are mainly carried by Cl − , the predicted E IPSC was − 36 mV.However, electrical stimulation of the inhibitory fibre tracts leaving the MNTB, the major source of glycinergic inhibition in the superior olivary complex (Fig. 6A,B), reliably elicited pharmacologically isolated glycinergic IPSCs with E IPSCs of at least − 70 mV in wild type KCC2 +/+ and homozygous KCC2 AD/AD mice.The driving force of the IPSCs, or else the strength of inhibition, is governed by the difference between the resting (in our case holding) potential and the reversal potential of the IPSC.IPSCs of homozygous KCC2 AD/AD neurons reversed at more hyperpolarized membrane potentials compared to wild type neurons, suggesting a larger driving force for inhibition in homozygous KCC2 AD/AD mice and thus increased KCC2 activity (Fig. 6C,D).These more hyperpolarizing IPSCs resulted in a leftward shift of the current-voltage relationship in both LSO and SPN neurons (Fig. 6E,F), which changed from − 72.0 ± 2.6 mV (n = 11) in LSO wild type neurons to − 80.6 ± 2.7 mV (n = 12) KCC2 AD/AD neurons (two-tailed t-test: p = 0.034; Fig. 6G).A hyperpolarizing shift was also observed from − 82.0 ± 2.1 mV (n = 19) in wild type SPN neurons to − 92.8 ± 1.4 mV (n = 13) KCC2 AD/AD neurons (two-tailed t-test: p ≤ 0.001; Fig. 6G).Thus, the phospho-mimetic mutation at Ser 937 affects not only the developmental shift in immature auditory neurons, but also increases the driving force for inhibition in mature neurons.

Discussion
We generated a transgenic KCC2 Thr 934Ala /Ser 937Asp mouse, in which serine 937 is replaced by aspartate mimicking the phosphorylated state and analysed its role in the auditory brainstem.This approach was motivated by the observation that the phospho-mimetic mutation of serine 937 is among the most effective ways to increase KCC2 activity in a heterologous expression system 65 , and since the auditory brainstem shows peculiarities concerning synaptic inhibition, such as posttranslational activation of KCC2 to induce D/H shift 47,49 and the most negative E Cl− observed so far in neurons 54 .
A fundamental process during development of the nervous system is the shift from depolarizing to hyperpolarizing action of inhibitory neurotransmitters.This is often accompanied by an increase in the expression or increase in the activity of KCC2 33,[40][41][42][43][44]46 . In he auditory brainstem, especially in rat LSO neurons, the D/H shift (5) occurs during the first postnatal week 8,20,74,75,84 .Here, KCC2 is perinatally expressed, but transport-inactive 47,49 .This raises the question how KCC2 activation is achieved during development 47,49 .Based on previous studies, three possible scenarios can be envisaged, which might be causally related: increase in the oligomer/monomer ratio 49 , a shift from membrane rafts to non-membrane rafts 50 , and phospho-regulation 51,85 .
Here, we hypothesized that the potent phospho-site serine 937 plays an important role and, therefore, generated KCC2 Thr 934Ala /Ser 937Asp mice.These mice are viable and bred normally.They thus provide an excellent tool to explore in vivo the effect of phosphorylation on KCC2 activation and activity.By performing electrophysiological analyses in LSO neurons of acute brainstem slices, we showed that the developmental maturation of Cl -dependent inhibition is shifted to earlier stages in homozygous KCC2 Thr 934Ala /Ser 937Asp mice.These results are consistent with in vitro analyses, as the KCC2 Thr 934Ala /Ser 937Asp double mutant leads to activation of KCC2 in HEK293 cells.Such enhanced KCC2 activity lowers the [Cl − ] i and increases the strength of glycinergic inhibition.Thus, phosphorylation of Ser 937 is sufficient to induce KCC2 activity causing an earlier D/H shift in LSO neurons.
Similarly, (de)phosphorylation of Thr 906/ Thr 1007 or Ser 940 induce the onset of hyperpolarisation during development 86,87 .The decrease in phosphorylation of Thr 906 and Thr 100752,67 increased KCC2 activity 60 .In transgenic KCC2 Thr 906Ala /Thr 1007Ala mice, mimicking the dephosphorylated state of these two phospho-sites, an accelerated onset of hyperpolarisation in hippocampal neurons was observed, pointing to its importance for the D/H shift in this brain area 86 .In contrast, heterozygous KCC2 Thr 906Glu /Thr 1007Glu mice, mimicking a phosphorylated state, showed a delayed D/H shift in CA3 pyramidal neurons and hippocampal slices 87 .These mice showed increased ultra-sonic vocalization, seizure susceptibility and long-term abnormalities in social behaviour 87 in comparison to transgenic KCC2 Thr 906Ala/Thr1007Ala mice.In the latter, the dephosphorylation-mimicking status limits the onset and severity of seizure 64 .Ser 940 is another phosphorylation site highly phosphorylated in cultured hippocampal neurons in the first postnatal week 86 .This enhances the KCC2 activity in cultured hippocampal neurons 69,86,88 .The importance of Ser 940 phosphorylation is further corroborated by a delayed D/H shift in Ser 940Ala knock-in mice 86 .These mice suffer from profound social interaction abnormalities like in autism spectrum disorders 24,86 .Thus, phospho-regulation is a common mechanism to regulate KCC2 activity and to initiate the onset of hyperpolarisation.In view of its rich phospho-site tool box, it will be interesting to study in the future, whether individual phospho-sites have brain area-specific functions.The increasing number of site-specific KCC2 transgenic mice pave the way for such an analysis.
So far, it is unclear whether the stronger Cl -extrusion in the transgenic KCC2 AD/AD mice has an implication for the auditory function.Presently, our gramicidin-perforated patch-clamp recordings revealed a conspicuous early D/H shift in KCC2 AD/AD mice (hyperpolarizing Cl -gradient present at P3), suggesting early onset of KCC2mediated Cl -extrusion.This result implies a shorter depolarizing phase of inhibitory inputs onto LSO neurons during postmitotic differentiation.In this phase of neuronal maturation, GABA/glycine triggered depolarisation increase [Ca 2+ ] i , thus contributing to synaptic maturation during development of neuronal circuits 9 .In the early postnatal LSO, GABA/glycine synaptic inputs evoke depolarizing EPSPs which induce Ca 2+ transients spreading to dendritic processes and even trigger action potentials 8,15,20,89 .During the first postnatal week, such excitation likely facilitates NMDA-dependent developmental plasticity, thereby contributing to synaptic refinement of the MNTB-LSO projections 90 .It remains to be investigated whether a shorter depolarizing phase, caused by early KCC2 activity onset, affects the development of excitatory and inhibitory synapses on LSO or SPN neurons.The D/H shift in the auditory brainstem is staggered in time, occurring before postnatal day 1 (P1) in the SPN to about P12 (i.e. at hearing onset) in the MNTB (cochlear nucleus: 48,76 ; superior olivary complex: 8,47,74,91 ).While the mechanisms underlying cell-type-specific regulation remain to be elucidated, these differences point to the importance of precise Cl -homeostasis regulation for maturation of the brainstem circuit.
Another peculiarity of the auditory brainstem is an extremely negative E Gly specifically in mature SPN neurons 54 .This is important for the precise offset firing after sound termination 53 .The neuron-type-specific regulation of E Cl− in auditory brainstem neurons might be due to a different phosphorylation pattern of KCC2 that specifically attunes the strength of inhibition.Consistent with this notion, the phosphorylation status correlates with hyperpolarizing Cl − gradient, as shown for E GABA in adult hippocampal neurons of Thr 906Ala /Thr 1007Ala transgenic mice 64 .Our analyses of KCC2 AD/AD mice demonstrate a more negative E IPSC both in SPN and LSO neurons compared to KCC2 WT .However, the present data do not explain the E Cl− difference between SPN and LSO neurons, as the mutation caused a similar hyperpolarizing shift of about − 10 mV in both nuclei.Therefore, it is tempting to presume additional regulatory mechanisms rendering E Gly more negative in the SPN.In the future, phospho-proteomic studies or phospho-site specific immunohistochemistry will be helpful to investigate the different phosphorylation pattern of KCC2 in SPN and LSO neurons.What effect will the more negative reversal potential have in SPN neurons?Remarkably, SPN neurons show very stable, strongly hyperpolarized IPSC reversal potentials after the D/H shift has taken place, without further changes after hearing onset around P12 92 .This suggests that the crucial window for adjusting the strength of inhibition is indeed around the D/H shift.Behavioural effects are to be expected in difficult sound localization tasks as well as gap-detection or sound duration discrimination tasks.Indeed, a reduction of KCC2 activity results in reduced gap detection in a cellular paradigm 54 .This loss of temporal resolution is present in ageing, hearing loss, and neurodegeneration 54 .Our results suggest that phosphorylation of Ser 937 enhances KCC2 activity resulting in more negative reversal potential in SPN neurons, which in turn might enhance gap detection.Therefore, pharmaceuticals that mediate a neuronal-targeted increase in phosphorylation of Ser 937 may represent a novel therapeutic strategy to prevent for example age-related hearing loss.In a high-throughput screening the compound CLP257 and its carbamate prodrug derivate CLP290 were found as promising KCC2 agonist compounds that enhance the activity of KCC2 93,94 and increase the phosphorylation of Ser 94095 .However, a follow-up study did not confirm the effect of CLP257 on KCC2 activity 96 .Future studies should test whether this compound also has an influence on the phosphorylation of Ser 937 .
In conclusion, we showed that a phospho-mimetic mutation of serine 937 enhances KCC2 activity in the mammalian auditory brainstem.This shifted the onset of hyperpolarisation, i.e.E Cl− , to an earlier timepoint in development and rendered E IPSC in mature LSO and SPN neurons more negative.Thus, phospho-regulation of KCC2 is a potent way to increase its transport activity.As such, pharmaceuticals that directly interfere with phosphorylation of KCC2 may be a novel therapeutic strategy.

Construction of the expression clones
Site-directed mutagenesis of mouse KCC2a (NM_001355480.1) was performed according to the QuikChange mutagenesis system (Stratagene, Heidelberg, Germany) 65,66,97 .The sequence for the forward oligonucleotide for the generation of the KCC2 Thr 934Ala /Ser 937Asp mutant is CGG GAG ATC CAG AGC ATC GCA GAC GAG GAC CGG GGC TCC ATT CGGAG.The generated clone was verified by sequencing (LGC genomics, Berlin).

Cell culturing
For immunocytochemistry and measurement of K + -Cl − cotransporter activity, HEK293 cells were transiently transfected with the respective constructs, using Turbofect (Fermentas, Schwerte, Germany).Cells were seeded in a 6-well plate 24 h prior transfection.The DMEM medium was replaced four hours before transfection.150 µl Opti-MEM (Invitrogen, Karlsruhe, Germany), 6 µl Turbofect and the corresponding DNA amount were thoroughly mixed and incubated for 20 min at room temperature.The mixture was applied to the cells which were then shaken at 300 rpm for 10 min at room temperature.
For K + -Cl − cotransporter activity measurements, transfected HEK293 cells were plated at a concentration of 1 × 10 5 cells/well in a 0.1 mg/ml poly-L-lysine coated black-well 96 well culture dish (Greiner Bio-One, Frickenhausen, Germany) 24 h after transfection.Each transfected construct was plated out three times, and represents three technical replicates.For the generation of independent biological replicates, the constructs were transfected separately.At least five biological replicates were done.The remaining cells were plated on 0.1 mg/ml poly-L-lysine-coated glass coverslips.After ~ 18 h, coverslips were proceeded for immunocytochemical analysis to determine transfection rates, which were routinely between 20 and 30%.

Immunocytochemistry
For immunocytochemistry, all steps were performed at room temperature.HEK293 cells grown on poly-L-lysinecoated coverslips were fixated for 10 min with 4% paraformaldehyde in 0.2 M phosphate buffer.Afterward, the cells were washed three times with PBS before the blocking solution (2% bovine serum albumin and 10% goat serum in PBS) was applied for 30 min.Primary antibody solution (anti-KCC2 N1-12; 1:1000; Neuromab, California, USA) was added in carrier solution (0.3% Triton X-100, 1% bovine serum albumin, 1% goat serum in PBS) and incubated for 1 h.After washing three times with PBS the secondary antibody, which was conjugated to a fluorescent probe (Alexa Flour 488 goat anti-mouse; 1:1000; Thermo Fisher Scientific, Bremen, Germany) was added to the carrier solution and incubated for 1 h.Again, the cells were washed three times with PBS and completely dried.The dried coverslips were mounted onto glass slides with Mowiol (Roth) and 4′,6-diamidine-2-phenylindole (DAPI, 1:1000; Roth).Photomicrographs were taken using an Olympus fluorescence microscope (Olympus BX63).

Determination of the K + -Cl -cotransport activity
Transport activity of KCC2 was determined by Cl − -dependent uptake of Tl + in HEK293 cells as described previously 72,73,98  Statistical Analysis: Transport activities of the respective mutant were tested against control samples (mmKC-C2a WT and mock), using a two-sample t-test after Student's t-test for similar variances between samples.In exceptional cases, where the standard deviation differs by more than a factor of 2, Welch's t-test was used 99 .To avoid pseudo-replication, the number of degrees of freedom was deflated according to the size of independent preparations each with three technical replicates.The false-discovery rate was controlled and p-values were corrected using the Benjamini-Hochberg method 100 .The chance of false positive results (type 1 errors) was reduced by choosing p-values < 0.01.
The distribution of genotypes in the F1 generation was tested with the χ 2 test under the null hypothesis of a Mendelian ratio of 1:2:1.The age-dependent weight (P0-P16) was tested with repeated measurement two-way ANOVA against the null hypothesis of no difference in mean weight, using sex, genotype, and the interaction of sex and genotype as fixed factors and the mouse id as random factor, i.e., aov(weight ~ sex*genotype + Error(id)).

Animals
All protocols were in accordance with the German Animal Protection Law and approved by the local animal care and use committee (LAVES (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit), Oldenburg) and followed the NIH guide for the care and use of laboratory animals.Mice of both sexes were used.The KCC2 AD/AD mouse line was generated with CRISPR/Cas (clustered, regulatory interspaced, palindromic repeats (CRISPR)-associated (Cas) system) at the transgenic core facility at the Max Planck Institute of Molecular Cell Biology and Genetics (Dresden, Germany).For this purpose the guide RNA 5′cucguagugucugcucagag 3′ and the repair template: CAT CCA CTG TAG TAA TGG CTC TTG GCA GGG CGT GGG TGG TGA CCC CCC AGC AGA GCT GGC ACC AAC CTG TGT CAC TCC CCA GAT CCA ATCG ATTGCCGATGAAGAC CGAGGC TCC ATT CGG AGGAA was used.These mice were kept on a C57Bl/6N genetic background.Primers for genotyping KCC2 wild-type are as follows : for : ATG GGC CCT TGA AGG ACA GG and rev : CCG AGA CTC GTC TGT GAT GCT.For the genotyping of the T934A/S937D mutation in KCC2 the following primers were used: for: ATG GGC CCT TGA AGG ACA GG, and rev: TCG GTC TTC ATC GGC AAT CGA.

Morphometric volume analysis
Adult KCC2 AD/AD and KCC2 +/+ mice (aged between postnatal day (P) 30-P60) were transcardially perfused with phosphate buffered saline (PBS; 136.9 mM NaCl, 2.7 mM KCl, 10.1 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4) followed by 4% paraformaldehyde solution.Brains were dissected and postfixed for 2-4 h in 4% PFA and stored in 30% sucrose in PBS for cryoprotection.The brainstem was cut in coronal sections with a thickness of 30 µm using a cryostat (Leica Biosystems, Wetzlar, Germany).Nissl staining was performed on consecutive sections of the auditory brainstem.Sections were imaged with an automated slide scanning microscope (AxioScan Z1, Carl Zeiss, Oberkochen, Germany).The volume of individual nuclei was obtained as described previously [101][102][103] .Shortly, images were analysed by outlining the auditory nuclei using Fiji 104 and multiplying this area with the thickness of each section.Three animals per genotype were analysed.The experimentalist was blind to the respective genotype.Statistical analysis was performed using Student's t-test after testing for Gaussian distribution using Prism version 9 (GraphPad, San Diego, California).Reported values are mean ± SD.

Figure 1 .Figure 2 .
Figure 1.Evolutionary conservation of phosphorylation site in vertebrate KCC isoforms.Multialignment of a C-terminal part of vertebrate KCCs were generated using ClustalW in Genious.Amino acid residues encoded by exon 22 are marked by a black bar.Exon 22 is only present in all vertebrate KCC2 and non-therian KCC4 isoforms.Phosphorylation sites that enhance KCC2 activity upon dephosphorylation are marked with blue asterisks.These phosphorylation sites are Thr 906 , Thr 1007 and Thr 1009 .They are highly conserved among all vertebrate KCC isoforms.Phosphorylation sites that are marked with green asterisks enhance KCC2 activity upon phosphorylation.These sites are Ser 932 , Thr 934 , Ser 937 , and Ser 940 .They are only present in KCC isoforms encoded by exon 22. hs (Homo sapiens), mm (mus musculus), rn (Rattus norvegicus), md (Monodelphis domesticus), gg (Gallus gallus). https://doi.org/10.1038/s41598-023-48884-x mutation of KCC2 at Ser 937 causes an early D/H shift of E Cl− compared to the control (− 79.4 mV vs. − 36.6 mV at P3, ***p < 0.001), suggesting an important role of this phospho-site in developmental regulation of KCC2.

Figure 4 .
Figure 4.No morphological changes in the auditory brainstem of homozygous KCC2 Thr 934Ala /Ser 937Asp mice.(A) Representative images of Nissl-stained sections of the auditory brainstem nuclei of adult homozygous KCC2 WT (KCC2 +/+ ) and KCC2 Thr 934Ala /Ser 937Asp (KCC2 AD/AD ) mice.Quantitative morphometric analyses revealed no differences in volume of the nuclei of the CNC (B) as well as of the SOC (C) between the two genotypes.Depicted are mean values ± SD, N = 3 mice/genotype; scale bar = 200 µm.CNC, cochlear nucleus complex; DCN, dorsal cochlear nucleus; LSO, lateral superior olive; MNTB, medial nucleus of the trapezoid body; SOC, superior olivary complex; SPN, superior paraolivary nucleus; VCN, ventral cochlear nucleus.

Figure 5 .
Figure 5. Earlier D/H shift in homozygote KCC2 Thr 934Ala /Ser 937Asp mice.Gramicidin perforated recording in LSO neurons.(A) (up) image of gramicidin perforated recording, visible patch pipette loaded with Atto 488; (below) whole-cell recording configuration with Atto488 inside the cell following patch rupture.(B) Representative traces from recordings made in control (KCC2 +/+ ) and homozygous KCC2 Thr 934Ala /Ser 937Asp (KCC2 AD/AD ) group at P3 showing current traces in response to glycine application at different holding potentials.(C) Representative plot of peak current amplitudes at different voltages for both groups at P3 (black control, red KCC2 AD/AD ).Line intersection with x-axis indicates the E Cl− .(D) Population data from control and KCC2 AD/AD at P3 and P5 showing significantly hyperpolarized E Cl− at P3 in KCC2 AD/AD compared to control.The control group shows a developmental change towards hyperpolarized values from P3 to P5.

Figure 6 .
Figure 6.Hyperpolarized E Cl− in mature LSO and SPN neurons of homozygous KCC2 Thr 934Ala /Ser 937Asp mice.(A) Coronal slice at the level of the superior olive showing the bipolar stimulation electrode (left) in the MNTB and a patch pipette (right) in the LSO.(B) Low magnification image of an LSO neuron filled with dye via the patch pipette.(C), (D) Synaptically evoked IPSCs recorded in LSO neurons of wild type (KCC2 +/+ ) (C) and homozygous KCC2 Thr 934Ala /Ser 937Asp (KCC2 AD/AD ) mice (D) over a range of holding potentials (− 120 to − 65 mV).(E), (F) The mean I/V relationship for the IPSCs in wild type neurons (black) and KCC2 AD/AD neurons (red) revealed a left shift towards more hyperpolarized IPSC reversal potentials (= line intersection with x-axis) in the LSO (E) and the SPN (F).(G) This difference in reversal potentials was significant for mature neurons in the LSO and SPN. https://doi.org/10.1038/s41598-023-48884-xwww.nature.com/scientificreports/